All forms of life are capable of incorporating inorganic sulfate into organic molecules. Our research is concerned with the early steps in the utilization of inorganic sulfate. We plan to study the structure-function relationships of the key enzymes involved in sulfate activation and sulfate transfer. Major emphasis will be on ATP-sulfurylase and APS-kinase from Penicillium chrysogenum. The former enzyme has been purified to homogeneity and extensively characterized physically and kinetically. Future research will aim at identifying the amino acid residues at the active site and elucidating the chemical mechanism of the reaction. We also plan to initiate a comparative biochemistry study (at the molecular level) of the sulfate activating enzymes from other microorganisms, plants, and animal tissues. Other specific projects include studies on the enzymes involved in the bio-synthesis and degradation of choline-O-sulfate, and on the membrane transport system for inorganic sulfate in fungi. Some of the methods that will be employed include: (1) chemical modification of specific amino acid residues, (b) affinity labeling, (c) equilibrium ligand binding with native and chemically modified enzymes, and (d) analysis of steady-state kinetics.